CN203419927U - PDMS (polydimethylsiloxane) based three-dimensional cell co-culture model - Google Patents

Abstract

The utility model discloses a three-dimensional cell co-culture model based on PDMS. An opening communicating with the left culture pool and the right culture pool is arranged on the layer, and the left culture pool and the right culture pool are equipped with stoppers that fit the sides of the interlayer. The PDMS-based three-dimensional cell co-cultivation model of the utility model can establish a three-dimensional co-culture system of two kinds of cells on the same plane, can realize the dynamic observation of the interaction between the two kinds of cells, and can carry out cell migration, chemotaxis, invasion and growth The tube experiment is convenient for in situ or collecting cells for molecular biology detection, and overcomes the defect that vertical co-culture cannot rule out the interference of gravity. It can be used repeatedly and has the advantages of simplicity, good repeatability, cheapness and safety.

Description

Three-dimensional cell co-culture model based on PDMS

technical field

The utility model relates to a cell culture model, in particular to a three-dimensional cell co-culture model based on PDMS.

Background technique

In vitro cell co-culture (co-culture) is to mix and co-culture two kinds of cells (from the same tissue or from different tissues), so that the shape and function of one of the cells can be stably expressed and maintained for a long time . Cell co-culture technology can simulate the microenvironment generated in vivo, which is convenient for better observing the interaction between cells and cells, and between cells and the culture environment, as well as exploring the mechanism of action and possible targets of drugs, filling the gap between monolayer cell culture and Whole Animal Experimentation Divide.

This technology can mainly involve experiments and applications in the following aspects: 1. Cell-to-cell contact, causing direct contact of plasma membranes and producing extracellular matrix ECM, which contains interstitial collagen, fibronectin, proteoglycan And laminin and other components, thereby simulating the microenvironment similar to that in vivo, maintaining the three-dimensional configuration of cells and promoting the function of cells. 2. The interaction between cells, observe the changes in the functions, properties and growth behavior of cells after co-culture, such as the influence of the secretion of a certain cell on the gene expression of another cell, the "cell-to-cell interaction" Dialogue" etc. 3. To study the effects of drugs on cell morphology and function and related mechanisms, and provide important technical support for the development of new drugs.

The current common cell co-culture methods can be roughly divided into direct contact co-culture and non-direct contact co-culture. Direct contact co-cultivation means that under suitable conditions, two or more types of cells are inoculated in the same culture dish in a certain proportion and co-cultivated, so that the two cells are in direct contact and produce cytokines through paracrine and autocrine effects. etc. to interact. The disadvantage is that it is difficult to separate the two kinds of cells, and it is not convenient for observation and subsequent detection. Non-direct contact co-cultivation refers to the inoculation of two or more types of cells on different carriers, and then placing these carriers in the same culture environment without direct contact between different types of cells. Since the two kinds of cells are easy to separate, it is convenient for observation and does not affect the subsequent detection, mainly including the following technical methods: 1. Use the culture supernatant of one kind of cells (containing different growth factors) to co-culture with another kind of cells. Its operation is simple, but it cannot dynamically observe changes in cell movement. 2. Put the slide inoculated with one type of cell into the culture dish of another type of cell for co-cultivation. This method is simple to operate, but it cannot dynamically observe the interaction between cells and cell movement. 3. The two kinds of cells are successively inoculated in the same culture dish for vertical co-culture, and the sandwich co-culture method is separated by Matrigel or polycarbonate membrane in the middle. 4. Transwell co-cultivation method: apply a kind of permeable cup-shaped device, the bottom of the cup is covered with a permeable polycarbonate membrane (pore size 0.1-12.0um) and put the Transwell chamber into the culture plate , cell A is planted in the upper chamber, cell B is inoculated in the lower culture plate and the components in the culture medium can affect the cells in the upper chamber, so that the influence of the substances secreted or metabolized by cell B on cell A can be studied. The advantage of the above two methods is that they can simulate the growth structure hierarchy of cells in the in vivo environment, but their vertical structure cannot exclude the interference of gravity for the detection of cell movement and is not conducive to dynamic observation. 5. Cell co-cultivation based on microfluidic chip: According to the different needs of experiments, design a microfluidic chip with a special structure, so that two kinds of cells can be co-cultured in different culture pools of the same chip. Its advantages are simple operation, large scale, saving sample amount, etc., and can also eliminate gravity interference factors, but it is not conducive to collecting cells for molecular biological detection.

Compared with the traditional two-dimensional monolayer cell culture, three-dimensional cell culture (TDCC) refers to the co-cultivation of carriers with three-dimensional structure and different materials with various types of cells in vitro, so that the cells It can migrate and grow in the three-dimensional space structure of the carrier to form a three-dimensional cell-carrier complex. Three-dimensional cell culture is to cultivate cells in a certain extracellular matrix, and the extracellular matrix (ECM) protein acts as a growth scaffold, enabling cells to differentiate to produce a certain three-dimensional tissue-specific structure, thereby creating a cell growth environment The in vivo environment can be simulated to the greatest extent. At present, the biological materials used in three-dimensional cell culture mainly include: artificial cartilage, PET polyvinyl ester, fibrin scaffold, collagen scaffold, rotating bioreactor, microcapsule and hollow fiber, etc.

Although many different cell co-culture methods and three-dimensional cell culture methods are widely used, they are all limited by technical problems and have their own shortcomings and deficiencies. Function and cell three-dimensional growth and movement changes, collecting sufficient cells for subsequent molecular biology testing and other aspects of experimental needs.

Contents of the invention

The purpose of this utility model is to provide a simple structure, low cost, with two separated cell culture pools, convenient for three-dimensional dynamic observation of the shape and movement changes caused by the interaction between cells, and conducive to the collection of cells for molecular biological detection based on PDMS The three-dimensional cell co-culture model overcomes the shortcomings of the existing technology.

The three-dimensional cell co-cultivation model based on PDMS of the present utility model comprises a model body, a left culture pool and a right culture pool are arranged inside the model body, an interlayer is arranged between the left culture pool and the right culture pool, and an interlayer is arranged on the interlayer. There is an opening communicating with the left culture pool and the right culture pool, and the left culture pool and the right culture pool are all provided with stoppers fitted to the sides of the interlayer.

In the PDMS-based three-dimensional cell co-cultivation model of the present invention, the opening in the width direction is wide at the bottom and narrow at the top; the left and right culture pools are arranged symmetrically with respect to the interlayer.

The PDMS-based three-dimensional cell co-cultivation model of the present invention, wherein the height of the model body and the interlayer is 8-12mm; the opening is located in the middle of the interlayer, and the lower width of the opening is 2.5-3.5 mm, The upper width is 1.5-2.5 mm.

The PDMS-based three-dimensional cell co-culture model of the utility model can establish a three-dimensional co-culture system of two kinds of cells on the same plane, can realize the dynamic observation of the interaction between the two kinds of cells, and can carry out cell migration, chemotaxis, invasion and growth The tube experiment is convenient for in situ or collecting cells for molecular biology detection, and overcomes the defect that vertical co-culture cannot rule out the interference of gravity. It can be used repeatedly and has the advantages of simplicity, good repeatability, cheapness and safety.

Description of drawings

Fig. 1 is the structural representation of the specific embodiment of the present utility model;

Fig. 2 is a top view schematic diagram shown in Fig. 1;

Fig. 3 is a schematic sectional view of A-A shown in Fig. 1;

Fig. 4 is the schematic diagram of the structure shown in Fig. 2 after adding a block in the culture tank;

5 is a schematic diagram of the mold structure for making the PDMS-based three-dimensional cell co-culture model of the present invention;

FIG. 6 is a schematic top view of FIG. 5 .

Detailed ways

As shown in Figures 1, 2, 3, and 4: 1 is a model body in the shape of a revolving body, and there are two culture pools inside the model body 1, namely a left culture pool 3 and a right culture pool 4. Between the left culture pool 3 and the right culture pool 4, there is an interlayer 2 integrated with the model body 1, and an opening 8 communicating with the left culture pool 3 and the right culture pool 4 is provided on the interlayer 2. The opening 8 is located in the middle of the interlayer 2 , that is, the left culture pool 3 and the right culture pool 4 are arranged symmetrically with respect to the interlayer 2 . Each of the left culture tank 3 and the right culture tank 4 is equipped with a stopper 7 that fits on the side of the interlayer 2, and when the two stoppers 7 are abutted on the interlayer 2, a formed opening is formed at the opening 8. a gap. The above-mentioned opening 8 may be in the shape of a wide bottom and a narrow top in the width direction. The lower width 6 of the opening 8 is 3 mm, that is all can be between 2.5-3.5 mm, and the upper width 5 is 2 mm, that is all can be between 1.5-2.5 mm. The height of the model body 1 and the interlayer 2 is 10 mm, that is, between 8-12 mm.

The fabrication process of the three-dimensional cell co-culture model based on PDMS of the present invention is as follows:

The material of model body 1 is SYLGARD 184 silicone rubber, which is a two-component kit product composed of liquid components, including basic components and curing agent. The basic components and curing agent are completely mixed at a mass ratio of 10:1, and the consistency of the medium-viscosity mixture is similar to that of SAE 40 engine oil.

As shown in Figures 5 and 6: 10 is a mold body, which has a mold body cavity 11, and a symmetrical semicircular right inner mold 12 and a left inner film 15 are placed inside the mold body cavity 11, and the right inner film 12 has a The right intimal cavity 13, and the left intimal cavity 14 inside the left intimal cavity 15. There is a gap between the right inner mold 12 and the left inner membrane 15 . Mold body 10, right inner mold 12 and left inner film 15 are all stainless steel materials. The depth of the mold body cavity 11, the depth of the right inner mold 12 and the left inner film 15 are all 10 mm, that is, all can be between 8-12mm.

Pour the above-mentioned completely mixed materials into the inner cavity 11 of the mold, the right inner cavity 13 and the left inner cavity 14, and bake and solidify at 80°C for 2 hours to form the PDMS model of the present invention.

After cooling, the solidified PDMS model is peeled off from the mold body 10, the right inner mold 12 and the left inner membrane 15 embedded therein are removed, and the semicircular shapes formed in the right inner membrane cavity 13 and the left inner membrane cavity 14 are solidified. Finally, a ring-shaped PDMS model body 1 with two culture pools and a spacer 2 in between the two culture pools and two semicircular PDMS modules are formed.

The interlayer 2 in the middle of the model body 1 is cut off at the midpoint, and the above-mentioned opening 8 is trimmed.

Cut the two semicircular PDMS modules into cuboid modules of appropriate size to form two stoppers 7 .

The PDMS-based three-dimensional cell co-culture model of the utility model can establish a three-dimensional co-culture system of two kinds of cells on the same plane, can realize the dynamic observation of the interaction between the two kinds of cells, and can carry out cell migration, chemotaxis, invasion and growth The tube experiment is convenient for in situ or collecting cells for molecular biology detection, and overcomes the defect that vertical co-culture cannot rule out the interference of gravity. It can be used repeatedly and has the advantages of simplicity, good repeatability, cheapness and safety.

Use of the model of the present utility model: before use, the above-mentioned model is subjected to autoclaving and ultraviolet irradiation sterilization.

Experimental example 1: The three-dimensional cell co-culture model based on PDMS is applied to the co-culture of two kinds of cells in the horizontal direction and the interaction between the two is observed.

Put this model body 1 into the 6-hole plate and press it tightly so that the bottom is close to the bottom of the 6-hole plate, and two cuboid-shaped stoppers 7 are respectively close to the two sides of the opening 8 of the compartment 2 to form a In one space, inject 20ul Matrigel (BD Company, USA) into the space, incubate at 37°C for 1 hour to solidify, and take out the cuboid-shaped stoppers 7 on both sides after the Matrigel solidifies. At this time, two culture pools, namely the right culture pool 4 and the left culture pool 3, are separated by the solidified Matrigel.

One type of cell is inoculated into the left culture pool 3, and the other type of cell is inoculated into the right culture pool 4. The culture fluid of the two can pass through Matrigel, which can realize the co-cultivation of two kinds of cells in the horizontal direction and dynamic observation, in situ Detect and collect cells for assay.

Taking glomerular endothelial cells as an example, they were inoculated in the right culture pool 4, and the left culture pool 3 was empty. Under the light microscope, it can be seen that the cells in the culture pool of this model body are compared with the cells in the normal culture plate, and the growth status of the cells is the same , the appearance was the same, there was no difference in the expression of cytoskeletal protein F-actin detected by Real time-PCR, Western blot and immunofluorescence, and there was no difference in the results of apoptosis detection. Afterwards, the glomerular podocytes were inoculated in the left culture pool, and co-cultured with the glomerular endothelial cells in the right culture pool for 24 hours. The endothelial cell growth curve and PCNA test results indicated that the co-cultivation of podocytes in this model can significantly stimulate renal The proliferation of glomerular endothelial cells is consistent with the experimental results using the traditional Transwell system.

Experimental example 2: The model of the utility model is applied to the experiment that one kind of cell (or the components in the culture medium) affects another kind of cell's three-dimensional shape and movement.

The model placement and cell inoculation methods are the same as in Experimental Example 1. Inoculate one kind of cell (or a culture solution containing a certain component) into the culture tank on the left side, and inoculate another kind of cell into the culture tank on the right side, you can dynamically observe the effect of the former on the latter, and make the latter move into the Matrigel The influence of growth and migration, so as to observe the changes in the three-dimensional shape and movement of cells. It is suitable for experiments such as cell migration, chemotaxis, invasion, angiogenesis of vascular endothelial cells in vitro, and tumor cell invasion.

For example, glomerular endothelial cells were inoculated in the right culture pool 4 of this model, and normal culture medium, culture medium containing VEGF (50pg/ml) and podocytes were added to the left culture pool 3 respectively, and the renal glomerular endothelial cells in these three groups were observed. Changes of morphology, migration and tube formation of bulbous endothelial cells under light microscope. The results show that both VEGF and podocytes can significantly stimulate glomerular endothelial cells to migrate into Matrigel and form a tubular structure. The results are consistent with the experimental results using the traditional Transwell system, but the PDMS-based The three-dimensional cell co-culture model has the advantage of being more convenient for dynamic observation.

Claims (3)

1. A three-dimensional cell co-culture model based on PDMS, characterized in that it includes a model body (1), a left culture pool (3) and a right culture pool (4) are arranged inside the model body (1), and the left culture pool A compartment (2) is provided between the pool (3) and the right culture pool (4), and an opening (8) connecting the left culture pool (3) and the right culture pool (4) is provided on the compartment (2), Both the left culture tank (3) and the right culture tank (4) are provided with stoppers (7) fitted to the sides of the compartment (2). 2. The PDMS-based three-dimensional cell co-culture model according to claim 1, characterized in that: the opening (8) is in the shape of a wide bottom and a narrow top in the width direction; the left culture pool (3) and The right culture tank (4) is arranged symmetrically with respect to the compartment (2). 3. The PDMS-based three-dimensional cell co-culture model according to claim 2, characterized in that: the height of the model body (1) and the compartment (2) is 8-12mm; the opening (8) Located in the middle of the compartment (2), the lower width (6) of the opening (8) is 2.5-3.5 mm, and the upper width (5) is 1.5-2.5 mm.

Images